Currently there is a great interest in nanotechnology, the manipulation and study of matter at the nanometer scale (see a recent Science review, Nov. 24, 2000). There is a belief that, in the long term, nanotechnology will lead to useful devices or structures functioning on the nanometer scale. In recent years, the manipulation of small molecules and atoms has been demonstrated; for example the scanning tunnelling microscope tip has been used to write “IBM” with xenon atoms and also to perform local catalysis on a metal surface. The atomic force microscope (AFM) tip has also been used for so-called “dip-pen” nanolithography (Piner et al, Science 1999, 28, 3661), to write features by contact with gold surfaces using thiol chemistry, down to 10-20 nm feature size. However, these methods are not compatible with the use of biological molecules and not straightforward to implement. This is because they require very clean surfaces and careful control over the conditions of deposition.
To date, the only methods of note for manipulation of biological molecules are optical tweezers, the atomic force microscope (AFM) and microfluidics. Optical tweezers have been applied to biological molecules attached to micron-size beads. For example, tweezers have been used to stretch DNA molecules. The AFM has been used to bring one molecule on the tip into contact with a molecule on the surface. Both these methods are limited to manipulating single molecules one at a time (for a review, see Nature Reviews 2000, 1, 130).
Microfluidics, using patterned polymer films, has been used to flow biological molecules down channels for separation and analysis. It has been used on large single DNA molecules flowing along a channel, for molecule by molecule analysis (Science 2000, 294, 1536)). This method is limited in the range of possible applications and does not allow the opportunity to write in biological molecules.
Microcontact printing has also been used to create patterns of biological molecules on a surface by direct stamping. This is limited to one type of biological molecule and also has limitations in the feature size possible.
DNA and peptide arrays, provided on a single chip, are important tools in molecular biology. Their production, on a commercial scale, poses various problems.
The current methods for writing with biological molecules are based on spotting using a pipette or piezoelectric delivery in air. For example, in the manufacture of DNA arrays, the feature size is about 50-100 μm. The photolithographic method for DNA chip production developed by Affymetrix has a fundamental feature size limit, due to diffraction of about 250 nm. Moreover, this limit has not yet been realised due to inefficiencies in the chemistry.
Schreiber has recently described a simple protein array, obtained by spotting proteins onto aldehyde-functionalised glass slides (Science 2000, 289, 1760-1763). The spot resolution was 150 μm, and there were technical issues with keeping the protein solvated, and blocking off unreactive aldehydic sites. Thus, the manipulation and writing with biological materials lag well behind that of atoms and small molecules due to the lack of suitable methods.
Scanning ion conductance microscopy (SICM) is a form of scanning probe microscopy, to image the surface of living cells, e.g. at 50 nm resolution. The method is based on scanning a micropipette over the surface of a cell and using the ion current that flows to an electrode inside the pipette to maintain and regulate the distance from the surface.
As disclosed in WO-A-00/63736, when the sample-pipette distance is modulated, an additional modulated current is produced, which adds to the DC current. The modulated signal only becomes significant when the pipette is close to the sample. This provides a robust, reliable method of distance control and virtually eliminates problems in dc drift or changes in ionic strength.
Zhang et al, J Vac. Sci. Technology 1999, B17(2), 269-272, discloses that a micropipette can be used to process microcircuits and microstructures on a substrate, by SICM. It is limited to conducting surfaces and to electrochemical reactions. The micropipette tip-surface gap is controlled by the ion current, without distance modulation. If a smaller voltage is applied, then less deposition takes place, but this also affects the position of the pipette with respect to the surface; since the current would be smaller, the micropipette would move away from the surface to maintain constant current. Control is thus difficult if not impossible.